NLRP3 regulates alveolar bone loss in ligature‐induced periodontitis by promoting osteoclastic differentiation

NLRP3 inflammasome is a critical part of the innate immune system and plays an important role in a variety of inflammatory diseases. However, the effects of NLRP3 inflammasome on periodontitis have not been fully studied.


| INTRODUC TI ON
Periodontitis is a chronic inflammatory and infectious disease affecting the periodontal tissues around the teeth, characterized by periodontal attachment loss, bone resorption, and finally leading to tooth loss. 1 Periodontitis is one of the most common oral diseases, with a high incidence in adults. 2 It is noteworthy that the inflammation and the spread of bacterial components during periodontitis have a close relationship with various systemic diseases, including Rheumatoid arthritis (RA), cardiovascular diseases and cancer. 3 Inflammasomes are multi-protein complexes assembled by intracytoplasmic pattern recognition receptors (PRRs), which are an important part of the body's immune system. 4 Nucleotide binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is one of the most typical inflammasomes, which is composed of NLRP3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) and pro-caspase-1. Toll-like receptors (TLRs) are a group of PRRs, 5 which can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and activate nuclear factor kappa B (NF-κB), resulting the formation of pro-IL-1β and up-regulated NLRP3 expression. This is the first step of NLRP3 inflammasome activation. The second step leads to the cleavage of pro-caspase-1 to caspase-1, followed by the conversion of pro-IL-1β to IL-1β through assembling NLRP3, ASC and procaspase-1 into an inflammasome complex. [6][7][8] In recent years, NLRP3 inflammasome has attracted widespread attention due to its extensive function in inflammatory diseases. 9 With the in-depth research on the mechanism of NLRP3 inflammasomes, a variety of small molecule compounds have been found to act as inhibitors of NLRP3 inflammasome for the treatment of NLRP3-mediated inflammatory diseases, such as MCC950, CY-09, INF39. 10,11 Recently, more attention has been paid to the association between NLRP3 inflammasome and periodontitis. In a mouse periodontitis model, knock out of NLRP3 decreases Porphyromonas gingivalis-associated alveolar bone loss. 12 Consistent results also observed in human tissue, a research shows NLRP3 and IL-1β have been found highly expressed in human gingival tissues with severe chronic periodontitis. NLRP3 expression is up-regulated with the treatment of stimulus in many periodontal cell types, including macrophage, periodontal ligament fibroblasts (PDLFs) and periodontal ligament cells (PDLCs). 13,14 However, it is well known that NLRP3 is highly expressed in monocytes/macrophages, 15 which could further differentiate into osteoclasts under suitable conditions. Therefore, we intend to investigate whether NLRP3 can regulate periodontitis-related alveolar bone loss by affecting osteoclast differentiation.
In this study, we used ligature-induced periodontitis models of NLRP3 knockout mice (NLRP3 KO ) and their wildtype (WT) littermates to compare their alveolar bone phenotypes. We further used Lysm-Cre/Rosa nTnG mouse to trace the changes of Lysm-Cre + osteoclast precursors in ligature-induced periodontitis with or without MCC950 treatment. At last, we explored MCC950 as a potential drug for the treatment of periodontitis in vivo and in vitro.

| Animals and Ligature-induced periodontitis
NLRP3 KO mice generated in a C57BL/6J background were kindly provided by Professor Shuo Yang. 16 The LysM-Cre mouse line was obtained from Gem Pharmatech Company (Stock Number T003822) and the Rosa nTnG mouse line was obtained from the Jackson Laboratory (Stock Number 023035). Lysm-Cre/Rosa nTnG mice were obtained by crossing Lysm-Cre mice with Rosa nTnG mice. Experimental periodontitis was induced in 2-month-old WT, NLRP3 KO and Lysm-Cre/Rosa nTnG mice. Briefly, 5-0 silk ligature was tied around the right maxillary second molar and the other side was left untied to serve as the baseline control. MCC950 (MCE, Cat#HY-12815) or PBS was injected intraperitoneally (i.p) to mice (10 mg/kg) at day 0, 1 and 2 and every 2 days thereafter. Mice were sacrificed 10 days after placement of the ligature. All mice were bred and maintained in the SPF Laboratory Animal Center of Nanjing Medical University. All animal procedures were conducted in accordance with approved guidelines of the Committee of Nanjing Medical University for Animal Resources (Approval ID 1906018).

| Micro-computed tomography (micro-CT)
Maxillae were removed and dissected free of all soft tissues for micro-computed tomography (Micro-CT) as described. 17

| Histology and histochemistry staining
Isolated maxillae were fixed in 4% paraformaldehyde, and then decalcified in 14% EDTA. After dehydration, the maxillae were embedded in paraffin for paraffin sections or embedded in Tissue-Tek OCT compound for frozen sections. The paraffin sections were stained with haematoxylin and eosin (H&E), histochemically for tartrateresistant acid phosphatase (TRAP) or alkaline phosphatase (ALP) and analysed as described previously. 18 The frozen sections of Lysm-Cre/Rosa nTnG mice were mounted with Mounting Medium containing DAPI (Vector) and images were captured with a Leica DM4000 fluorescence microscope, as we reported previously. 19

| Immunohistochemistry staining
The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H 2 O 2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against IL-1β (R&D System, Cat#AF-401-NA). After rinsing with PBS for 15 minutes, tissues were incubated with secondary antibody (HRP-Donkey Anti-Goat IgG, Proteintech, Cat#SA00001-3) at room temperature.
Sections were then washed and colours were developed with DAB (3,3'-diaminobenzidine). Next, haematoxylin was used as a counterstain. Images were captured with a Leica DM4000 fluorescence microscope.

| ELISA
Cell supernatants were removed and analysed using ELISA kits according to the manufacturer's instructions to measure the release of IL-1β (Invitrogen, Cat#88-7013).

| Western blot analysis
The protein content of supernatants was concentrated using
After washing with PBS for 30 minutes, cells were incubated respectively with Donkey anti-Goat Cy3 (Beyotime, Cat#A0502) at room temperature. Cells were stained with DAPI and images were captured with a Leica DM4000 fluorescence microscope.

| Statistical analysis
All data are given as mean ± SD. Statistical analysis was performed using Graphpad prism 7 software (GraphPad Software Inc, San Diego, CA, USA). Comparisons between 2 groups were analysed using the 2-tailed unpaired Student's t-test. Comparisons among 3 or more groups were carried out using one-way ANOVA followed by Dunnett's post-hoc multiple comparisons. P values <.05 were considered statistically significant.

| NLRP3 deficiency protects against alveolar bone loss in ligature-induced periodontitis
NLRP3 inflammasome has been reported to be associated with periodontitis closely. [20][21][22] To explore the role of NLRP3 in periodontitis, we used a classic periodontitis model, ligature-induced periodontitis.
Two-month-old NLRP3 KO mice and their WT littermates were used and 10 days after placement of the ligature, the mice were sacrificed.
μCT and histomorphometric analyses were performed to observe al- Furthermore, TRAP staining was performed to evaluate osteoclast bone resorption. TRAP-positive osteoclast surface was reduced in ligature-induced periodontitis from NLRP3 KO mice than that from WT mice ( Figure 1E,F). ALP staining was next performed to evaluate F I G U R E 1 NLRP3 deficiency protects against alveolar bone loss in ligature-induced periodontitis. Two-month-old NLRP3 KO mice and their WT littermates were used. 5-0 silk ligature was tied around the right maxillary second molar and the other side was left untied to serve as the control group. All the mice were sacrificed 10 d  Figure 1E-H). At last, flow cytometry confirmed that the percentage of CD11b + Gr-1 −/low osteoclast precursors was lower in ligature-induced periodontitis from NLRP3 KO mice than that from WT mice ( Figure 1I,J).

| NLRP3 deficiency prevents IL-1β release and inhibits osteoclast differentiation
OCPs from NLRP3 KO and WT mice were used to determine the influences of deletion of NLRP3 gene on inflammatory response.
After treatment with LPS and Nigericin, activated caspase-1 p20 (an auto-processed fragment of caspase-1) and cleaved IL-1β were increased in OCPs from WT mice (Figure 2A). Similarly, ELISA data showed the release of IL-1β in OCPs from WT mice ( Figure 2B).
However, neither activated caspase-1 nor cleaved IL-1β was observed in OCPs from NLRP3 KO mice under the same conditions ( Figure 2A,B).
To determine if NLRP3 inflammasome directly affects osteoclast differentiation, we cultured OCPs from both NLRP3 KO and WT mice with M-CSF and RANKL. To activate NLRP3 inflammasome, cells were exposed to LPS and Nigericin during osteoclast formation. We found that OCPs from NLRP3 KO mice formed less osteoclasts than OCPs from WT mice, with or without stimulus ( Figure 2C). Additionally, the expression levels of osteoclast-related genes, including Ctsk, Acp5, Oscar and Atp6v0d2, were all significantly decreased in OCPs from NLRP3 KO mice compared with those from WT mice ( Figure 2D). Interestingly, LPS-primed OCPs from WT mice formed more osteoclasts and expressed higher levels of osteoclast-related genes in the presence of Nigericin, but this effect was not observed in OCPs from NLRP3 KO mice ( Figure 2C,D).
Given the markedly increased level of cleaved/released IL-1β in NLRP3 activated OCPs (Figure 2A Figure 2E, OCPs from WT mice receiving IL-1β neutralizing antibody plus LPS and Nigericin formed less osteoclasts than LPS and Nigericin-treated OCPs. However,similar results did not appear in OCPs from NLRP3 KO mice ( Figure 2E). Therefore, NLRP3 may mediated osteoclast differentiation through IL-1β production.  Figure 3A). Importantly, MCC950 partially blocked the increased level of IL-1β in LPS plus Nigericin-treated OCPs ( Figure 3A).

| MCC950 decreases osteoclast differentiation
To further determine whether MCC950 targets are NLRP3 or other molecules, the effects of MCC950 on NLRP3 inflammasome activation were detected in OCPs from NLRP3 KO mice and their WT littermates. The protein levels of caspase-1 p20, cleaved IL-1β and released IL-1β were evidently reduced in supernatants from MCC950treated WT OCPs ( Figure 3B,C). By contrast, the protein levels of caspase-1 p20, cleaved IL-1β and released IL-1β were undetectable in supernatants from MCC950-treated NLRP3 KO OCPs ( Figure 3B,C).
In order to observe the effect of MCC950 on osteoclast differentiation intuitively, OCPs were cultured with M-CSF and RANKL.
TRAP staining showed that both LPS-primed and Nigericin-stimulated promoted osteoclast differentiation in OCPs from WT mice, but MCC950 played the opposite role ( Figure 3D). Similar trends appeared in real-time RT-PCR, which was used to examine the expression levels of osteoclast-related genes including Ctsk, Acp5, Nfatc1 and Oscar ( Figure 3E). Importantly, MCC950 could not exert the similar effects on OCPs from NLRP3 KO mice ( Figure 3D,E), suggesting MCC950 may decrease osteoclast differentiation through NLRP3 inflammasome inhibition.

| MCC950 reduces the number of Lysm-Cre + osteoclast precursors in ligature-induced periodontitis
We next used Lysm-Cre/Rosa nTnG mouse, a double-fluorescent Cre reporter mouse, to observe the effects of MCC950 on Lysm-Cre + osteoclast precursors in vivo. In Lysm-Cre/Rosa nTnG mouse, all Lysm-Cre + cells and their descendants express GFP, whereas all Lysm-Cre − cells express tdTomato. 26 We examined the number of Lysm-Cre + cells in ligature-induced periodontitis and control group from Lysm-cre/Rosa nTnG mice receiving MCC950 or vehicle by frozen embedded sections of maxilla. It was clear that more Lysm-Cre + cells were found in ligature-induced periodontitis than control group ( Figure 4A-C). Notably, MCC950-treated periodontitis group reduced the number of Lysm-Cre + cells compared with vehicle-treated periodontitis group ( Figure 4A-C).
Proteins from periodontal tissues around maxillary second molar were extracted and examined for the expression level of IL-1β, which represents the activation state of NLRP3 inflammasome.
We found that the expression level of IL-1β was increased in periodontitis group compared with that in control group. Importantly, the expression level of IL-1β was decreased in MCC950-treated group compared with that in vehicle-treated group, as expected ( Figure 4D,E).

| MCC950 inhibits Lysm-Cre + osteoclast precursors differentiation into osteoclast
To further determine the effects of MCC950 on osteoclast differentiation, Lysm-Cre-GFP + cells from bone marrow were sorted out by flow cytometry after treated with MCC950 or vehicle ( Figure 5A). First, Lysm-Cre-GFP + cells were subjected to osteoclastogenic assay with M-CSF and RANKL. TRAP staining was performed to observe osteoclast formation ( Figure 5B). Both TRAP + osteoclast area and TRAP + osteoclast number were decreased clearly from the mice receiving MCC950 compared with that receiving vehicle ( Figure 5B,C). Besides, Lysm-Cre-GFP + cells were also used to test the expression levels of osteoclast-related genes by real-time RT-PCR. The expression levels of genes, including Ctsk, Acp5, Nfatc1 and Atp6v0d2, were appreciably downregulated from the mice receiving MCC950 compared with that receiving vehicle ( Figure 5D).

| MCC950 protects against alveolar bone loss in ligature-induced periodontitis by inhibiting the NLRP3 inflammasome activation and osteoclast differentiation
To

| D ISCUSS I ON
The main findings of the present study are as follows ( Thereafter, another well-done and extensive study used ageing-related periodontitis as an in vivo model, suggested that NLRP3 plays an important role in osteoclastogenesis during ageing. 29 However, ageing is a very complex process. Besides NLRP3 mediated inflammation, osteoclast differentiation in ageing has been affected by many other factors. Therefore, in our study, we used ligature-induced periodontitis models of NLRP3 KO  is mainly expressed in monocytes/macrophages, 35 it is also found in mesenchymal stem cells 36 and osteoblasts. 37 Up-regulation of NLRP3 in mesenchymal stem cells inhibits osteogenic differentiation. 38 In osteoblasts, MCC950 inhibits NLRP3 inflammasome-mediated pyroptosis and promotes early osteogenic differentiation. 39 Correspondingly, our data also found that NLRP3 KO mice had increased osteoblast activity in ligature-induced periodontitis, compared with WT mice. Thus, it is possible that the increased alveolar bone volume caused by NLRP3 deficiency may be partly due to the effects of NLRP3 on osteoblast differentiation. In the future, we may generate myeloid specific NLRP3-overexpression F I G U R E 6 MCC950 protects against alveolar bone loss in ligature-induced periodontitis by inhibiting the NLRP3 inflammasome activation and osteoclast differentiation. Two-month-old WT mice received i.p. injection of MCC950 or vehicle were used. 5-0 silk ligature was tied around the right maxillary second molar and the other side was left untied to serve as the control group. All the mice were In summary, by using ligature-induced periodontitis murine models, we have demonstrated that both NLRP3 deficiency and MCC950 reduce the number of osteoclast precursors and prevent osteoclast differentiation, thereby protect against alveolar bone loss in ligature-induced periodontitis. Thus, our results suggest that NLRP3 regulates bone resorption in periodontitis by mediating osteoclast differentiation. MCC950 may be used as a potential drug to treat periodontitis in the future.